Abstract
Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a critically important regulatory lipid of the plasma membrane (PM); however, little is known about how cells regulate PM PI(4,5)P2 levels. Here, we show that the phosphatidylinositol 4-phosphate (PI4P)/phosphatidylserine (PS) transfer activity of the endoplasmic reticulum (ER)-resident ORP5 and ORP8 is regulated by both PM PI4P and PI(4,5)P2. Dynamic control of ORP5/8 recruitment to the PM occurs through interactions with the N-terminal Pleckstrin homology domains and adjacent basic residues of ORP5/8 with both PI4P and PI(4,5)P2. Although ORP5 activity requires normal levels of these inositides, ORP8 is called on only when PI(4,5)P2 levels are increased. Regulation of the ORP5/8 attachment to the PM by both phosphoinositides provides a powerful means to determine the relative flux of PI4P toward the ER for PS transport and Sac1-mediated dephosphorylation and PIP 5-kinase-mediated conversion to PI(4,5)P2. Using this rheostat, cells can maintain PI(4,5)P2 levels by adjusting the availability of PI4P in the PM.
| Original language | English |
|---|---|
| Pages (from-to) | 1797-1813 |
| Number of pages | 17 |
| Journal | Journal of Cell Biology |
| Volume | 217 |
| Issue number | 5 |
| DOIs | |
| Publication status | Published - May 1 2018 |
| Externally published | Yes |
Bibliographical note
Funding Information:2 d after transfection, cells were lysed with 200 最l/well of 1 䬀 Lae- mmli buffer, and lysates were gently denatured at 65°C for 30 min Acknowledgments in Eppendorf tubes before SDS-PAGE. 40 最l of each sample was We are grateful for DNA constructs provided by the Grinstein, loaded onto 4–12% Tris-glycine mini-gel (Thermo Fisher Scien-Meyer, Yin, and Varnai laboratories. Confocal imaging was per-tific). After SDS-PAGE at 110 V in Tris-glycine SDS running buffer formed at the Microscopy and Imaging Core of the National (Thermo Fisher Scientific), proteins were transferred to nitrocel-Institute of Child Health and Human Development, National lulose membranes (0.45 µm pore size; Thermo Fisher Scientific) Institutes of Health, with the kind assistance of Dr. Vincent at 100 V for 1 h in Tris-glycine transfer buffer (Thermo Fisher Sci-Schram. We also thank Dr. Joshua Pemberton for critical reading entific) containing 5% methanol (vol/vol). Nonspecific antibody of the manuscript. binding was blocked with Odyssey Blocking Buffer (LI-COR) for This work was supported in part by the intramural research 1 h at room temperature. Primary antibodies were diluted in 5 ? program of the Eunice Kennedy Shriver National Institute of BSA-PBS solution (wt/vol) and incubated at 4°C overnight. Pri-Child Health and Human Development at the National Insti-mary antibodies were diluted in the following ratio: α-tubulin tutes of Health. The work of J.P. Zewe, R.C. Wills, and G.R.V. 1:2,000, α-OSBPL5 1:500, and α-ORP8 1:500. After three washes Hammond was supported by National Institutes of Health (for 10 min each) with 0.1% Tween 20–containing PBS solution grant 1R35GM119412-01 (to G.R.V. Hammond). The work of (vol/vol), fluorescence-conjugated secondary antibodies (diluted 1:5,000) were incubated in PBS-containing 5% BSA at room temperature for 1 h. Fluorescence signal was detected with the Odyssey 9120 imaging system (LI-COR), and the images obtained from individual wavelengths were presented with grayscale by using OdysseyV3.0 software.
Funding Information:
J. Humpolickova, L. Vrzal, D. Chalupska, V. Veverka, and E. Boura was supported by a grant from the Czech Science Foundation (17-05200S, to E. Boura) and by the Academy of Sciences of the Czech Republic (RVO:61388963). G.D. Fairn was supported by a Canadian Institutes of Health Research open operating grant (MOP-133656). The authors declare no competing financial interest.
Publisher Copyright:
© 2018 Crown.
ASJC Scopus Subject Areas
- Cell Biology